In order to understand the latency of human immunodeficiency virus (HIV- 1) infection and the effect of various cofactors, several experimental models have been developed. Although cells of liver origin are not yet considered as target tissue for HIV-1 infection, they can serve as an important model, since hepatic abnormalities and a high incidence of hepatitis B virus infection are found with AIDS. We observed that a clone of human hepatoblastoma HepG2 cells are CD4 positive and can be infected with HIV-1 and support HIV-1 replication by producing infection virions. In contrast to the stimulatory effect of tumor necrosis factor (TNF-alpha) or Phorbol-12 myristate-13-acetate (PMA) in HIV-1 replication in T cells, these agents inhibited HIV-1 replication in liver cells. The long-term objective and specific aims of this proposal are the following: 1) Determine the mechanisms(s) involved in inhibition of HIV-1 infection by TNF-alpha in HepG2 cells. Compare the effect of TNF-alpha with that of PMA on HIV-1 infection in this system, which may indicate a novel pathway for HIV-1 infection. 2) Compare these data with other hepatoma cell lines including the HepG2 clone which is CD4 negative. 3) Analyze the state of the HIV-1 DNA and RNA in these infected cells. 4) Isolate and characterize TNF-alpha induced gene sequences by subtraction hybridization of HepG2 derived cDNA libraries. Similarly subtracted cDNA clones will be isolated from PMA treated HepG2 cells as well. These cDNAs will be cloned in an eukaryotic expression vector so that they can be transfected into various cell lines including HepG2 to obtain stable cell lines for analysis of their resistance to HIV-1 infection. 5) In addition to the strain III B, we will use HIV-1 proviral clone pHXBc2 for infection. Various stable cell lines containing this proviral genome will be isolated. The effect of TNF- alpha or PMA will be evaluated in these infectious clones derived from various hepatoma cell lines. The role of protein kinase C in relation to PMA effect will be studied. 6) Gel retardation assay and extensive DNA footprinting will be used to identify the proteins in TNF-alpha or PMA treated cells which bind to the regulatory regions of HIV-1. 7) By mutating various regions of this proviral clone, we intend to localize the region(s) in HIV-1 genome responsible for the TNF-alpha or PMA mediated inhibition. 8) We will identify, characterize, purify, and if required, isolate the cDNAs encoding the trans-acting proteins from various hepatoma cell lines that bind to HIV-1 LTR by screening a lambdagt11 library and compare with that of treated cells. 9) The binding of TNF-alpha and PMA treated HepG2 nuclear protein to HIV-1 TAR RNA sequences will be studied.